We plan to: 1) Determine the complete kinetic mechanism of yeast inorganic pyrophosphatase by measuring the rates of a) water-phosphate exchange, b) phosphate-pyrophosphate exchange, both on and off the enzyme, and c) solvent isotope effects. Studies will be conducted as a function of phosphate concentration, pH, and the nature of the divalent metal-ion cofactor. Structural analogs of phosphate and pyrophosphate will be utilized to explore enzyme specificity. 2) Locate within the covalent structure the apparently essential carboxylic acid side chain revealed by chemical modification studies. Attempts will also be made to repeat the findings of the Avaeva group, implicating an aspartyl side chain as the site of phosphorylation in a putative phosphoryl enzyme intermediate. If these attempts are successful, the localization of this residue will also be carried out. 3) Determine the remaining unknown distances of interest between the two metal-ion and two phosphate sites on the enzyme. 4) Structurally characterize the stable enzyme:Mg2ion:PPi:F- complex. 5) Obtain cryosolvents suitable for the study of the enzyme at low temperature. The methods to be employed include 31p and 19F nmr, Mn2ion and Co2ion epr, rapid-mixing quenched-flow, CNBr and proteolytic digestion, peptide fingerprinting, amino acid analysis.